Decreased muscarinic receptor binding in subjects with schizophrenia: a study of the human hippocampal formation.

BACKGROUND: Acetylcholine is important to hippocampal function, including the processes of learning and memory. Patients with schizophrenia show impaired learning and memory and hippocampal dysfunction. Thus, acetylcholinergic systems may be primarily or secondarily disrupted in the hippocampal formation of schizophrenic patients. The present study tested the hypothesis that [(3)H]pirenzepine-labeled muscarinic cholinergic receptor levels are altered in the hippocampal formation of patients with schizophrenia. METHODS: We have used quantitative autoradiography to measure [(3)H]pirenzepine binding to M(1) and M(4) receptors in the hippocampal formation from 15 schizophrenic and 18 nonschizophrenic subjects. RESULTS: The mean density of [(3)H]pirenzepine binding was reduced in all regions studied, including the dentate gyrus, subdivisions of Ammon's Horn (CA1-CA4), subiculum, and the parahippocampal gyrus, of the schizophrenic cohort. Moreover, unlike controls, there was no significant variation between the mean levels of [(3)H]pirenzepine binding across the subregions of the hippocampal formation from schizophrenic subjects. CONCLUSIONS: These findings provide support for a possible involvement of the muscarinic cholinergic system in the pathology and/or treatment of schizophrenia.     

Muscarinic1 and 2 receptor mRNA in the human caudate-putamen: no change in m1 mRNA in schizophrenia

Studies using tissue obtained at autopsy suggest that changes in cholinergic neurons could be important in the pathology of schizophrenia.1-4 We have previously reported a decrease in [3H]pirenzepine binding5 and [3H]AF-DX 384 binding6 to caudate-putamen (CP) from subjects who had schizophrenia. Under the conditions chosen, [3H]pirenzepine would predominately bind to muscarinic1 (M1) and muscarinic4 (M4) receptors,7whereas [3H]AF-DX 384 would mainly bind to muscarinic2 (M2) and M4 receptors.8 Given the relative concentrations of M1, M2 and M4 receptors in the human CP and the magnitude of the decreases in radioligand binding in schizophrenia, our results most likely reflected a change in the density of M1 and M2 receptors in the CP from the schizophrenic subjects. In situ hybridisation has now been used to determine levels of m1 and m2 mRNA in CP from 14 schizophrenic and 16 control subjects previously used for radioligand binding. m2 mRNA in the CP from the schizophrenic and control subjects was below the sensitivity of in situhybridisation. There was no difference in the levels of m1 mRNA in CP from schizophrenic and control subjects (mean +/- SEM: 103 +/- 16 vs106 +/- 17 fmol [35S]oligonucleotide probe g-1estimated tissue equivalents, P = 0.91). In conclusion, data from our radioligand binding studies show decreases in [3H]pirenzepine binding that are likely to reflect a decrease in the density of M1 receptors in CP from schizophrenic subjects. Our data in this study show the absence of a concomitant change in mRNA coding for that receptor.     

Effects of serotonergic denervation on the density and plasticity of brain muscarinic receptors in the rat.

This study investigated whether serotonergic lesion may affect density, sensitivity, and plasticity of muscarinic receptors in hippocampus and cerebral cortex. Intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) in rats produced a 90% reduction in cortical and hippocampal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents. In these brain areas, the 5,7-DHT lesion did not affect the overall density of muscarinic receptors or those of M1 and non-M1 muscarinic receptor subtypes as assayed using [3H]N-methylscopolamine ([3H]NMS), [3H]pirenzepine, and [3H]NMS in the presence of pirenzepine, respectively. In addition, the binding of the muscarinic agonist [3H]oxotremorine-M (OXO-M), taken as an indirect index of coupling efficiency of non-M1 receptors with G-proteins, did not change significantly in cortex and hippocampus of 5,7-DHT-lesioned rats. Similarly, carbachol-induced accumulation of [3H]inositol phosphates (InPs) in hippocampal miniprisms showed no significant differences between tissues from 5,7-DHT-lesioned and sham-operated rats. In sham-operated rats, an intraperitoneal (i.p.) injection of scopolamine (10 mg/kg once daily) during 21 days caused an increased density of [3H]NMS binding sites in cortex (+20%) and hippocampus (+26%). This up-regulation was restricted to non-M1 receptors subtypes. In 5,7-DHT-lesioned rats, chronic scopolamine failed to modify significantly the density of cortical or hippocampal M1 or non-M1 receptors. These results suggest 1) that 5-HT denervation did not affect the density and sensitivity of muscarinic receptors and 2) that the ability of cortical and hippocampal non-M1 receptors to up-regulate following repeated injection of scopolamine requires the integrity of 5-HT neurons terminating in these brain structures.     

Decreased muscarinic1 receptors in the dorsolateral prefrontal cortex of subjects with schizophrenia

To test the hypothesis that muscarinic receptors are involved in the pathology of schizophrenia, we measured muscarinic(1) (M1R) and muscarinic(4)(M4R) protein and mRNA as well as [(3)H]pirenzepine binding in Brodmann's areas (BA) 9 and 40 obtained postmortem from 20 schizophrenic and 20 age/sex-matched control subjects. There was a significant decrease in [(3)H]pirenzepine binding to BA 9 (mean +/- SEM: 151 +/- 15 vs 195 +/- 10 fmol mg(-1) ETE; P< 0.02), but not BA 40 (143 +/- 13 vs 166 +/- 11 fmol mg(-1) ETE), from subjects with schizophrenia. The level of M1R protein (0.11 +/- 0.007 vs 0.15 +/- 0.008 OD; P < 0.01), but not M4R protein, was decreased in BA9 from schizophrenic subjects with neither receptor protein being altered in BA 40. The level of M1R mRNA was decreased in BA 9 (30 +/- 7.0 vs 79 +/- 14 dpm x 10(3) mg(-1) ETE, P < 0.01) and BA 40 (28 +/- 5.9 vs 99 +/- 14, P < 0.01) with schizophrenia but M4R mRNA was only decreased in BA 40 (48 +/- 6.6 vs 89 +/- 9.9, P < 0.005). These data suggest that the M1R, at least in the dorsolateral prefrontal cortex, may have a role in the pathology of schizophrenia.     

Alterations of muscarinic and GABA receptor binding in the posterior cingulate cortex in schizophrenia

The posterior cingulate cortex (PCC), a key component of the limbic system, has been implicated in the pathology of schizophrenia because of its sensitivity to NMDA receptor antagonists. Recent studies have shown that the PCC is dysfunctional in schizophrenia, and it is now suspected to be critically involved in the pathogenesis of schizophrenia. Studies also suggest that there are abnormalities in muscarinic and GABAergic neurotransmission in schizophrenia. Therefore, in the present study we used quantitative autoradiography to investigate the binding of [(3)H]pirenzepine, [(3)H]AF-DX 384 and [(3)H]muscimol, which respectively label M1/4 and M2/4 muscarinic and GABA(A) receptors, in the PCC of schizophrenia and control subjects matched for age and post-mortem interval. The present study found that [(3)H]pirenzepine binding was significantly decreased in the superficial (-24%, p=0.002) and deep (-35%, p<0.001) layers of the PCC in the schizophrenia group as compared with the control group. In contrast, a dramatic increase in [(3)H]muscimol binding was observed in the superficial (+112%, p=0.001) and deep layers (+100%, p=0.017) of the PCC in the schizophrenia group. No difference was observed for [(3)H]AF-DX 384 binding between the schizophrenia and control groups. The authors found a significant inverse correlation between [(3)H]pirenzepine binding in the deep cortical layers and [(3)H]muscimol binding in the superficial layers (rho=-0.732, p=0.003). In addition, negative correlations were also found between age and [(3)H]pirenzepine binding in both superficial and deep cortical layers (rho=-0.669 p=0.049 and rho=-0.778, p=0.014), and between age of schizophrenia onset and [(3)H]AF-DX 384 binding (rho=-0.798, p=0.018). These results for the first time demonstrated the status of M1/M4, M2/M4 and GABA(A) receptors in the PCC in schizophrenia. Whilst the exact mechanism causing these alterations is not yet known, a possible increased acetylcholine and down regulated GABA stimulation in the PCC of schizophrenia is suggested.     

Autoradiographic comparison of muscarinic M1 and M2 binding sites in the CNS of spontaneously hypertensive and normotensive rats

Spontaneously hypertensive rats (SHR) respond with exaggerated pressor responses of central origin in response to pharmacologic stimulation of brain muscarinic receptors when compared with those to normotensive Wistar Kyoto (WKY) rats. At least part of the enhanced response to central muscarinic stimulation may be due to alterations in the expression of one or more of the five subtypes of muscarinic receptors. SHR are also known to exhibit regional alterations in the levels of mRNA encoding the M1, M2 and M4 receptors. In this study, we estimated the number of specific muscarinic receptor binding sites in 12-week-old SHR and WKY by measuring the binding of M1- and M2-selective ligands. Using standard autoradiographic techniques, coronal sections obtained from 12-week-old SHR and WKY were incubated with [³H]pirenzepine or [³H]AFDX 384 to label M1 and M2 receptors, respectively. Although both strains exhibited similar distribution patterns for both binding sites, sections derived from SHR expressed a significant increase in the number of [³H]pirenzepine binding sites compared to normotensive WKY in caudate putamen, CA3 region of the hippocampus, cingulate cortex, substantia nigra, posterior hypothalamic area and tuberomammillary nucleus. An increased number of [³H]AFDX 384 binding sites in SHR were observed in the olfactory tubercle, nucleus accumbens, basolateral amygdaloid nucleus, rostroventrolateral medulla and nucleus paragigantocellularis. Decreases in the number of [³H]AFDX 384 binding sites in SHR were also observed in the parietal cortex, medial geniculate, and lateral hypothalamic area. Statistically significant site-selective differences in binding densities between strains ranged from 4.0% to 35.5% of WKY means. These alterations in the expression of M1 and M2 binding sites in cardiovascular regions may contribute to the strain's hyper-responsiveness to cholinergic drugs and possibly to the appearance of other autonomic or behavioral phenotypes exhibited by SHR, including the hypertensive state itself.     

Hippocampal muscarinic supersensitivity after AF64A medial septal lesion excludes M1 receptors

Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of [3H]QNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, [3H]hemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (Bmax) in non-M1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M1 subtype.     

Low muscarinic receptor binding in prefrontal cortex from subjects with schizophrenia: a study of Brodmann's areas 8, 9, 10, and 46 and the effects of neuroleptic drug treatment

OBJECTIVE: Aberrant cholinergic inputs and synaptic neurotransmission in the prefrontal cortex induce cognitive impairment, which is a central feature of schizophrenia. Postsynaptic excitatory muscarinic cholinergic M(1) and M(4) receptors are the major cholinoceptive targets in the prefrontal cortex and hence may be involved in the pathology and/or pharmacotherapeutics of schizophrenia. METHOD: Using quantitative autoradiography, the authors analyzed the binding of the M(1)/M(4) receptor selective antagonist [(3)H]pirenzepine in prefrontal cortex (Brodmann's areas 8, 9, 10, and 46) from schizophrenia patients who had (N=6) or had not (N=11) been treated with the anticholinergic agent benztropine mesylate and from normal comparison subjects (N=20). Moreover, preliminary studies of [(3)H]pirenzepine binding in rat frontal cortex following administration of antipsychotic drugs or benztropine mesylate were performed. RESULTS: Relative to those of comparison subjects, the mean levels of [(3)H]pirenzepine binding were significantly lower in Brodmann's areas 9 and 46 of the schizophrenia patients not treated with benztropine mesylate (18% lower in Brodmann's area 9 and 21% lower in Brodmann's area 46) and in all four examined regions of the patients who had received benztropine (51%-64% lower). Antipsychotic or anticholinergic drugs tended to increase or have no effect on the density of [(3)H]pirenzepine-labeled receptors in rat frontal cortex. CONCLUSIONS: Because M(1) and M(4) receptors are critical to the functions of prefrontal cortical acetylcholine, the present findings suggest a functional impairment in cholinergic neurotransmission in schizophrenia and the possibility that muscarinic receptors are involved in the pharmacotherapeutics of the disorder.     

Normalization of subtype-specific muscarinic receptor binding in the denervated hippocampus by septodiagonal band grafts

Unilateral fimbria-fornix lesions were made by aspiration in female Sprague-Dawley rats. In a group of these rats, fetal septal tissue was transplanted into the lesion cavity. Lesion of the fimbria-fornix resulted in a reduction of cholinergic input to the hippocampal formation as indicated by the loss of acetylcholinesterase (AChE)-positive staining in all ipsilateral hippocampal laminae and a loss of [3H]hemicholinium-3 binding to cholinergic terminals in the strata oriens (82% reduction) and radiatum (77% reduction) of areas CA2 and CA3 and in the molecular layer of the dentate gyrus (83% reduction). In contrast, the density of muscarinic receptor binding ([3H]QNB) increased in the strata oriens (80% increase) and radiatum (70% increase) in areas CA2-CA4. This was shown to be due to an actual increase in receptor number (Bmax) and not to a change in affinity (KD). Analysis of muscarinic receptor subtypes indicated that the increase in receptor binding in the stratum radiatum was of the M-1 subtype ([3H]-pirenzepine) and in the stratum oriens was of the M-2 subtype ([3H]QNB + 100 nM pirenzepine). In the host hippocampus after fetal septal graft, the staining for AChE, the binding of [3H]hemicholinium-3, and the binding of muscarinic receptors (both the M-1 and M-2 receptor subtypes) were all comparable to nonlesioned control values. These data indicate that the fetal septal grafts have reinnervated the host hippocampus and have made synaptic contact with host cells in a manner capable of regulating postsynaptic muscarinic receptors.     

Decreased density of muscarinic receptors in the superior temporal gyrusin schizophrenia

Recent studies have indicated that muscarinic receptors are involved in the pathophysiology in schizophrenia, particularly in cognitive deficits. The superior temporal gyrus (STG) is an area that has also been strongly implicated in the pathophysiology of schizophrenia. Therefore, in this study, we investigated the binding density of two muscarinic antagonists, [(3)H]pirenzepine and [(3)H]AF-DX 384, in the STG of schizophrenia patients compared with controls. A significant decrease (44% in the superficial layers and 48% in the deep layers, P<0.01) in binding density of [(3)H]pirenzepine was observed in schizophrenia patients, which suggested a reduction of muscarinic M1 and M4 receptor densities in the STG of schizophrenia patients. A tendency toward decreased [(3)H]AF-DX 384 binding density (34%, P=0.09) was also observed in schizophrenia patients compared with controls. Because of the positive correlation between [(3)H]pirenzepine and [(3)H]AF-DX 384 binding, and, insofar as both ligands have high affinities for the M4 receptor, the involvement of M4 receptor alteration is also suggested in the STG in schizophrenia. These results suggest that changes of the muscarinic receptors M1 and M4 might contribute to the STG pathology in schizophrenia.     

Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland.

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]diphenyl-acetoxy-N-methyl-piperidine ([3H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25 degrees C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [3H]QNB and [3H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M1, M3 subtypes selective antagonist) > atropine (general) > pirenzepine (M1). Methoctramine (M2) didn't antagonise the [3H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca2+ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M1 and M3 muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.     

Human M1-, M2- and M3-muscarinic cholinergic receptors: binding characteristics of agonists and antagonists

The muscarinic acetylcholine receptors were identified in membrane preparations from human tissues by the specific binding of 1-[benzilic-4,4'-3H] quinuclidinyl benzilate. Saturation binding isotherms of this radioligand yielded a total amount of receptors of 435 +/- 208, 159 +/- 65 and 913 +/- 89 fmol/mg protein, respectively, in the hippocampus, pons and submandibular gland. Non linear least squares analysis of competition binding studies with the antagonists pirenzepine and AF-DX 116 indicates that the majority of receptors are of the M1-type in the hippocampus (83%, high affinity for pirenzepine, intermediate affinity for AF-DX 116), the M2-type in the pons (low affinity for pirenzepine and high affinity for AF-DX 116), and the M3-type in the submandibular gland (low affinity for pirenzepine and AF-DX 116). Competition binding parameters of the agonists carbachol, arecoline, oxotremorine, pilocarpine and MCN-A-343 were compared for M1, M2 and M3 receptors in the human hippocampus, pons and submandibular gland. GTP caused a shift to the right and a steepening of the shallow agonist competition curves in the 3 tissues but did not affect the initially steep ones. This effect is explained by a GTP-mediated conversion of high- to low-agonist affinity sites. The extent of the nucleotide shift was much greater for M2 receptors as compared with M1 and M3 receptors. The GTP effect was impaired by the sulphydryl reagent N-ethylmaleimide, probably due to alkylation of GTP-binding proteins. Moreover, the reagent provoked also an increase of the agonist affinity for the uncoupled muscarinic receptors. For all agonists, this increase was more pronounced for the M2 receptors than for the M1 and M3 receptors. These findings suggest structural differences between the agonist binding sites of M1 and M3 receptors versus the M2 receptors.     

M2/M4 muscarinic receptor binding in the anterior cingulate cortex in schizophrenia and mood disorders

We have previously shown a decrease in [(3)H]pirenzepine binding to M1/M4 muscarinic receptors in the anterior cingulate cortex in schizophrenia but not in major depression or bipolar disorder. The present study aimed to extend these findings by examining the binding of [(3)H]AF-DX 384 to M2/M4 receptors in the same cohort of subjects. Using quantitative autoradiography we measured [(3)H]AF-DX 384 binding in the anterior cingulate cortex of 15 schizophrenia, 15 bipolar, 15 major depression and 15 control cases. Post-mortem tissue was obtained from the Stanley Foundation Brain Bank. [(3)H]AF-DX 384 binding had a homogenous distribution amongst the layers of the anterior cingulate cortex, was higher in males than in females and declined with prolonged storage of tissue. An inverse correlation between [(3)H]AF-DX384 binding and age of onset of the disease was observed in the schizophrenia group suggesting that the earlier the age at onset the higher the binding was. In the depression group, there was a significant effect of gender on [(3)H]AF-DX 384 binding with females having lower binding in comparison to males. In the bipolar group, there was a significant inverse correlation between antipsychotic medication and [(3)H]AF-DX 384 binding, suggesting that the higher the dose of medication the lower the binding was. No differences in [(3)H]AF-DX 384 binding were seen between the four groups. The present results provide no evidence of M2/M4 receptor alterations in the anterior cingulate cortex in schizophrenia and affective disorders and extend the body of evidence implicating cortical M1 but not M2 involvement in the pathology and pharmacotherapy of schizophrenia.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

A pentylenetetrazole-induced generalized seizure in early life enhances the efficacy of muscarinic receptor coupling to G-protein in hippocampus and neocortex of adult rat

We have previously shown that exposure to the anti-cholinesterase eserine provokes interictal-like discharges in the CA3 area of hippocampal slices from adult rats in which a generalized seizure has been induced by pentylenetetrazole (PTZ) when immature (at 20 days). Such increased responsiveness to acetylcholine (ACh) was not associated with any change in hippocampal acetylcholine or gamma-aminobutyric acid (GABA) content, GABAergic inhibition or density of ACh innervation, but was blocked by the muscarinic receptor antagonist atropine. We therefore turned to quantitative radioligand binding autoradiography, in situ hybridization and the [35S]GTPgammaS method to assess the properties of hippocampal and neocortical muscarinic receptors in adult rats having experienced a PTZ seizure at P20. The densities of M1 and M2 receptor binding sites, respectively labeled with [3H]pirenzepine and [3H]AFDX-384, as well as the amount of m1, m2 and m3 receptor mRNAs, did not differ from control in the hippocampus and neocortex of these rats. In contrast, in PTZ rats, both brain regions displayed a marked increase in [35S]GTPgammaS incorporation stimulated by ACh, bethanechol and particularly oxotremorine. This finding indicates that a generalized seizure in immature rat can entail a long-term and presumably permanent increase in the efficacy of G-protein coupling to muscarinic receptors in the hippocampus and neocortex of the adult. By analogy, such a mechanism could account for the susceptibility to epilepsy of human adults having suffered from prolonged convulsions in early life.     

Distinct kinetic binding propeties of N-[3H]-methylscopolamine afford differential labeling and localization of M1, M2, and M3 muscarinic receptor subtypes in primate brain

Three classes of muscarinic receptors in mammalian brain have been postulated on the basis of equilibrium and kinetic binding data. However, equilibrium binding assays alone have not permitted a clear demonstration of the localization of putative M1, M2, and M3 receptor subtypes in the brain because of the overlaping affinities of virtually all muscarinic antagonists. In the present study, the conditions for selective occupancy of the M1, M2, and M3 receptor subtypes in the brain of the rhesus monkey were based on the distinct kinetic and equilibrium binding properties of N-[³H]-methylscopolamine (NMS) at cloned m1–m4 muscarinic receptor subtypes expressed in A9L transfected cells. Quantitative autoradiography of the M1, M2, and M3 muscarinic receptor subtypes in the primate brain was performed according to the following strategy. The M1 (m1) receptor subtype was labeled directly with a non-saturating concentration of [³H]-pirenzepine. The M2 (m2) subtype was labeled by incubations consisting of short, two minute pulses of [³H]-NMS after a preincubation with 0.3 μM pirenzepine to occlude m1, m3, and m4 sites. Selective occupancy of the M3 (m3) receptor (subtype) was achieved by pre-incubation with 0.5 nM unlabeled NMS to partially occlude the m1, m2, and m4 sites, equilibrium with 0.5 nM [³H]-NMS, followed by a 60 minute tracer dissociation in the presence of 1 μM atropine. In vitro autoradiography demonstrated that the M1 receptor subtype was confined to forebrain structures. M1 receptors were prevalent throughout the cerebral cortical mantle, amygdala, hippocampus, and the striatum. Low to background levels of the M1 receptor subtype were measured over the thalamus, hypothalamus, and brainstem. The M2 subtype was widely distributed with elevated densities of binding sites seen over all primary sensory cortical areas, and within discrete thalamic, hypothalamic, and brainstem nuclei. The distribution of the M3 receptor subtype was largely coincident with the pattern of the M1 sites labeled by non-saturating concentrations of [³H]-pirenzepine with some notable exceptions. Within the cerebral cortical mantle, the M3 receptor exhibited an elevated gradient over the orbitofrontal gyrus and the temporal lobe. Within the striatum, the M3 subtype was elevated over the anterior and dorsal part of the caudate nucleus, while the M1 receptors were most prevalent over the ventromedial sector. Selective labeling of M3 receptors was seen over the medial division of the globus pallidus and within the substantia nigra pars reticulata. In contrast to the pattern of the M1 receptor subtype, M3 receptors were prevalent also over midline nuclei of the hypothalamus. These results demonstrate that the distinct kinetic and equilibrium binding profiles of N-methylscopolamine and pirenzepine for cloned muscarinic receptors provide a viable ligand autoradiographic strategy for mapping the distribution of M1, M2, and M3 receptors in brain. © 1993 Wiley-Liss, Inc.     

Selectivity of pirenzepine in the central nervous system. I. Direct autoradiographic comparison of the regional distribution of pirenzepine and carbamylcholine binding sites

The binding capacities of the novel antagonist pirenzepine and the agonist carbamylcholine were examined autoradiographically to compare their abilities to reduce the binding of 1-[3H]quinuclidinyl benzilate ([3H]-1-QNB). This technique, which is applicable to any muscarinic ligand, permits a direct comparison between the binding of carbamylcholine and pirenzepine in the same assay. Analysis of the binding curves generated by standard scintillation counting of whole-brain slices indicated that the ligands bound heterogeneously to muscarinic receptors in the brain. Following apposition of slides to tritium-sensitive film, the binding profile for each ligand was examined visually and by microdensitometry. Regional analyses indicated that the agonist carbamylcholine displayed highest potency for thalamic nuclei, lower potency for cortical regions, and the lowest affinity for layers of the hippocampus. The M1-selective ligand pirenzepine displayed the highest potency for the dentate gyrus of the hippocampus, with lower inhibition levels in the cortex, and the lowest levels of inhibition found in the thalamus. The distribution of high affinity agonist sites was found to be distinct from the distribution of high-affinity antagonist binding sites. In a separate assay, the regional inhibition of pirenzepine and scopolamine was compared for the hippocampus and the forebrain. While scopolamine did not distinguish between muscarinic receptor sites in the hippocampus and cortex, pirenzepine inhibited [3H]-1-QNB labeling in the hippocampus significantly greater than in the cerebral cortex, providing additional evidence for the hypothesis that pirenzepine is a selective antagonist.     

Calcium antagonist, adenosine A1, and muscarinic bindings in rat hippocampus after transient ischemia

The protective roles of Ca2+ channel blockers against ischemic hippocampal damage are still debated. We used autoradiography to study postischemic L-type Ca2+ channels (1,4-dihydropyridine Ca2+ channel blocker binding), adenosine A1 receptors, and muscarinic cholinergic receptors in the rat hippocampus using [3H]PN200-110 (PN), [3H]cyclohexyladenosine (CHA), and [3H]quinuclidinyl benzilate (QNB), respectively, in 49 rats subjected to 20 minutes of forebrain ischemia. The rats were decapitated after 1 (n = 7), 3 (n = 7), 6 (n = 8), 12 (n = 7), 24 (n = 6), 48 (n = 6), or 168 (n = 8) hours of recirculation; eight control rats were sham-operated but experienced no cerebral ischemia. Reduced receptor binding preceding the delayed death of CA1 pyramidal cells was first observed in the stratum oriens of the CA1 subfield. Significant reductions in [3H]PN, [3H]CHA, and [3H]QNB bindings of this stratum compared with control were noticed after 3 (35%, p less than 0.01), 12 (31%, p less than 0.01), and 1 (10%, p less than 0.05) hours of recirculation, respectively. By 168 hours after ischemia (when the populations of CA1 pyramidal cells were depleted) all strata in the CA1 subfield had lost most of their receptor sites, and [3H]PN, [3H]CHA, and [3H]QNB bindings in the stratum oriens were decreased to 23%, 30%, and 63% of control (p less than 0.01). Although [3H]PN binding in the CA3 subfield did not change significantly during 168 hours after ischemia, the histologically intact dentate gyrus exhibited a 31% loss of binding sites compared with control (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)